Hfdpc

In vitro cytotoxicity and cell proliferation studies on normal hair follicle dermal papilla cells were undertaken as described previously [33 (link)]. Cultured human hair follicle dermal papilla cells (HFDPCs) (Promo-Cell, Heidelberg, Germany) from passage 4 were seeded (7.5 × 10⁴ or 3.75 × 10⁴ per mL) onto a 96-well plate (Nunc, Wiesbaden, Germany) and grown to a confluence of 60–70% for 24 h. The MTT assay for cytotoxicity and proliferation was conducted for 5 days. Treatment was conducted with dutasteride alone (dutasteride was dissolved in DMSO at 100 mM and serial dilutions were prepared from 100 to 1.6 µM), dutasteride in NLCs uncoated or coated with 5% CSO-LA (dutasteride concentration 12.5–100 µM), and empty NLCs uncoated and coated with CSO-LA, with minoxidil (100 µM) as a positive control. Absorbance was determined spectrophotometrically at 570 nm using a microplate reader (SpectraMax^®M2e, Mol. Devices, San Jose, CA, USA). The results are expressed as percentages of untreated controls in four cultures. The reported values are the means ± SD.

BCE powder, CaNZac-35, was purchased from Koyo Mercantile Co. (Tokyo, Japan). BCE contained high concentrations of anthocyanins (38.0 g/100 g BCE) [11 (link)]. HFDPCs derived from the temples of Caucasian middle-aged women were obtained from PromoCell (Heidelberg, Germany). Cells were maintained in follicle dermal papilla cell growth medium (PromoCell). All cell culture experiments were conducted at 37 °C in a humidified incubator containing 5% CO₂.

Human follicle dermal papilla cells (HFDPCs) were purchased by PromoCell. The cell line was cultured in a specific medium (follicle dermal papilla cell growth medium-ready to use with its supplement mix, PromoCell) in conditions of complete sterility and maintained at 37 °C with 5% carbon dioxide (CO₂) atmosphere. The product HYDRO DELUXE BIO was weighed and dissolved at the concentration of 80 mg/ml in a complete cell culture medium.

To evaluate the effect of the Cicaria fraction on the expression of hair growth factors, a genetic analysis of the hair growth factors was conducted in HFDPCs. First, HFDPCs were purchased from PromoCell (Heidelberg, Germany) and cultured in a DPC growth medium containing the supplement and 100 U/mL of penicillin and 100 ug/mL of streptomycin. For gene expression analysis, HFDPCs were inoculated in a 6-well plate at a density of 3 × 10⁵, and then cultured for 24 h in an incubator at 37 °C and 5% CO₂. After 24 h, the culture medium was discarded, washed with DPBS, and replaced with a medium not containing supplements or a medium containing 10 ppm Minoxidil, as a positive control, or Cicaria fraction 1 ppm, and further cultured for 24 h. Subsequently, RNA was isolated from cells using an RNeasy Mini Kit (Qiagen); RNA was quantified at 260 nm using nanodrop, and the cDNA was synthesized in amplifiers (C1000 Thermal Cyclers, Bio-Rad, USA) using 2 μg of RNA. The degree of VEGF, FGF7, and HGF gene expression was finally evaluated by performing a real-time polymerase chain reaction in a real-time PCR machine using a mixture of FGF7 and HGF primers and cyber green (SYBR Green Supermix) to synthesize cDNA. Primer sequences and reaction conditions are shown in Table 1, and the expression levels of genes were analyzed relative to the β-actin gene.